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JMed Genet 1991; 28: 159-162
159
Molecular studies of non-disjunction in trisomy 16
T J Hassold, D Pettay, S B Freeman, M Grantham, N Takaesu
Abstract
The origin of the additional chromosome in 26
trisomy 16 spontaneous abortions was studied using
DNA probes for chromosome 16, including a probe
for centromeric a satellite sequences. We were able
to determine the parent and meiotic stage of origin
of trisomy in 22 cases, with all being attributable to
maternal meiosis I non-disjunction. Furthermore,
in each of the remaining four cases the results were
compatible with this origin. Thus, it is likely that
the high incidence of trisomy 16 results from an
abnormal process acting at maternal meiosis I
which more frequently involves chromosome 16
than other similar sized chromosomes. In studies of
recombination, we found little evidence for an
association between reduced or absent recombination and chromosome 16 non-disjunction; however,
we were unable to rule out an effect of hyperrecombination.
Materials and methods
The study population consisted of 26 trisomy 16
abortuses, 12 of which were ascertained in a cytogenetic survey of spontaneous abortions conducted in
Hawaii and 14 in a similar study now under way at
Emory University, Atlanta, GA. Preliminary results
on the 12 Hawaiian cases were reported previously2;
an additional three cases described in that report were
excluded from the present study because of insufficient proband DNA. The methodology for the
collection, culturing, and cytogenetic analysis of
tissue samples is almost identical for the Hawaii and
Atlanta studies, and has been described in detail
elsewhere.3
DNA was extracted from fetal tissue and parental
blood samples and processed for DNA hybridisation
studies using previously described techniques.4 The
parental origin of the additional chromosome was
evaluated with eight probes detecting restriction
fragment length polymorphisms at chromosome 16
loci: pEKMAD2. 1 (D16S83), CMM65 (D16S84),
Trisomy 16 is the most commonly identified trisomy 16/32 (D16S35), 16/12 (D16S36), hp2alpha (HP),
in man, occurring in at least 1% of all clinically pEKXp3B (CTRB), p79-2-23 (D16S7), and
recognised pregnancies.' Despite its high frequency, pHUAPRT (APRT).5
little is known about the parent or meiotic stage of
The meiotic stage of origin of trisomy was studied
origin of the additional chromosome. Results of with pSE16-2 (D16Z2), which detects highly polychromosomeheteromorphism analyseshaveimplicated morphic a satellite sequences at the centromere of
maternal non-disjunction as the most likely source of chromosome 16.6 For this analysis, DNA samples
trisomy,' but these studies have been hindered by the from the trisomic fetus and parents were digested
imprecision of the cytogenetic technique and the low with EcoRV and processed for hybridisation studies
level of polymorphism associated with the chromo- as for the other probes. Non-disjunction was scored as
some 16 cytogenetic marker.
being of meiosis I origin if all centromeric restriction
We recently initiated DNA marker studies to fragments in the parent of origin were present in the
determine the parent and meiotic origin of the extra trisomic fetus, and of meiosis II origin if only a subset
chromosome in trisomy 16 spontaneous abortions and of the fragments were present in the fetus. This
to assess the possibility that hypo- or hyper-recom- scoring system assumes that the parent of origin is
bination is associated with the non-disjunctional heterozygous; however, this seems a safe assumption,
event. In the present report, we summarise our since (1) Greig et aP6 were unable to detect identical
observations on an initial series of 26 fetuses and show pSE16-2/EcoRV restriction patterns among approxithat virtually all trisomy 16 results from non- mately 30 unrelated persons, and (2) in the present
disjunction at maternal meiosis I.
study, in 22 of 23 cases in which the parental origin of
trisomy was determined, the trisomic fetus clearly
Division of Medical Genetics, Department of Pediatrics, inherited only a subset of the centromeric restriction
Emory University School of Medicine, 2040 Ridgewood fragments from the parent who had contributed a
Drive, Adanta, Georgia 30322, USA.
T J Hassold, D Pettay, S B Freeman, M Grantham, single chromosome 16.
For trisomies in which we were able to determine
N Takaesu
the
parent and meiotic stage of origin, we evaluated
Correspondence to Dr Hassold.
the frequency of crossing over between the two nondisjoined chromosomes by (1) identifying heteroReceived for publication 27 July 1990.
Revised version accepted for publication 10 September 1990.
zygous loci in the parent of origin of trisomy, (2)
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Hassold, Pettay, Freeman, Grantham, Takaesu
160
studying these loci in the trisomic offspring to was maternally derived and, as maternal heterozygosity
determine if heterozygosity had been maintained, or at two loci was maintained in the trisomic fetus, the
had been reduced to homozygosity, and (3) comparing trisomic line must have originated in meiosis.
the different loci in the trisomic offspring for evidence However, the centromeric marker was uninformative
of recombination. For example, if the parent of origin and therefore we were not able to distinguish between
a meiosis I and II error. In S9, we were unable to
were heterozygous at two loci and the trisomic
offspring heterozygous at one and homozygous at the determine the parental origin of trisomy, but all
other, a single (or odd number of) crossover(s) must parental restriction fragments detected by the centromeric probe were present in the fetus. Thus, the
have occurred between these loci.
additional chromosome 16 presumably resulted from
non-disjunction at meiosis I. In S374 and S382,
parental origin studies were uninformative. However,
Results
The results ofthe DNA marker studies are summarised in each case the fetus had inherited all of the maternal,
in table 1 and examples of determinations of parental but only a subset of the paternal, centromeric
and meiotic stage of origin of trisomy are shown in restriction fragments. Thus, for these two cases the
figs 1 and 2. We were able to determine both the trisomy must have originated from an error at
parent and meiotic stage of origin of non-disjunction maternal meiosis I or at paternal meiosis II.
Table 1 also summarises the results of our analyses
in 21 cases of simple trisomy 16 and in one case (S9)
with a 47,XX,+ 16/48,XX,+2,+ 16 chromosome of recombination between the non-disjoined chromoconstitution. In all 22 cases, the trisomy arose from somes. We detected recombination in 17 of the 22
cases involving maternal meiosis I non-disjunction; in
non-disjunction at maternal meiosis I.
We were also able to obtain some information on six of these we identified two crossovers and in the
each of the four remaining trisomies. In K3021, a other 11 cases one crossover. We were unable to
45,X/46,X,+16 mosaic, the additional chromosome detect recombination in the other five cases involving
Table I Summaty of DNA marker studies of 26 spontaneous abortions with trisomy 16.
Recombination studies at
different chromosome 16 locit
ID
No*
Chromosome
constitution
Parental ages
Fa
Mo
Origin of
trisomyt
16q
cen
16p
.,
q
C
t
N
N
Mat (1)
34
R
R
R
N
R
Mat I (2)
30
R
R
N
R R
Mat I (2)
35
N
R N
N
R
29
Mat I (3)
R
R
N N
N
Mat I (3)
30
N N
R
N
R
Mat 1 (2)
33
R R
N
N
29
Mat I (3)
N
R
31
Mat I (3)
R
N
N
R
Mat I (3)
29
R
N
R
R
N
22
Mat 1 (3)
N
N
N
Mat I (1)
32
R
R R
N
N
Mat 1(2)
36
N
N
? I
39
+16
S3
473,XY,
R
N
N
N
N
N
N
Mat
1
33
59
(2)
+2,+ 16
473,XX,±+16/48,XX,
R
N
N
N
Mat 1 (2)
33
34
S16
47,XY, +16
R R
R
N
R
Mat 1 (3)
30
31
47,XX,+16
S37
N
N
Mat
297(1)
30
47,XY,+ 16
S134
N
N
N
Mat
306(1)
30
47,XY,+ 16
S138
N
N
R
R
Mat 1 (2)
32
30
S145
47,XY, +16
R
R
N
N
28
30
Mat I (1)
S244
47wKXY,+ 16
N
I
Mat
29
29
16
(3)
S245
47,X,+
N
N
N
N
1
Mat
34
32
555
(2)
473,XX,+16
R
N
R
N
Mat I (5)
30
32
S343
47,-XY, +16
R
N
R
R
R
Mat I (1)
36
31
S356
47,XY,±+16
37
39
S374
47,XY, +16
36
31
47,XX, +16
S382
of spontaneous abortions in Honolulu; those with an S prefix were ascertained
in
a
were
a
K
with
study
cytogenetic
prefix
ascertained
*Samples,
in a similar study in Atlanta.
informative for parental origin of trisomy.
tNo in ( )=number inof markers
or R (reduced) if heterotFor loci heterozygous parent of origin, trisomic fetus is scored as N (non-reduced) if heterozygosity is maintained,
zygosity is converted to homozygosity. Crossing over must have occurred between adjacent loci whenever one is N and the other R.
K33021
K3029
K3056
K3074
K3086
K3164
K3259
S93361
K3375
K13407
K3409
K3425
45,X/46,X,+ 16
47,XX,+16
47,XX,+16
47,XY,+16
47,XY,+16
47,XX, + 16
47,XY,+ 16
47,#XY+16
47,XY,+16
47,XX,+16
47,XY,+16
47,XY,+16
34
42
37
34
32
35
33
34
28
23
35
37
28
35
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Molecular studies of non-disunction in trisony 16
161
0
0
0
ClI
co
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ILU Co
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IX Co
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LL
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o
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_E,
__'
*90
r.t
_- .
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W.
DNA marker studies
ofparental origin of trisomy 16.
DNA from three trisomy 16
spontaneous abortions (S37.0,
S134.0, S138.0) and their
parents was digested with TaqI
and probed with p79-2-23,
which detects multiallelic
variation at D16S7.7 Results are
consistent with maternal nonforS37.0 and S138.0
disjunction
since both have inherited a single
allele from the father and, by
dosage, two copies of a maternal
allel. An effect ofdosage is
evident also forS134.0; howver,
as the parents are heterozygous for
the same alleles, a parental origin
determination cannot be made.
Table 2 Evidence for recombination in maternally derived
sex chromosome trisomies, trisomy 16, and trisomy 21.
-C)
.EI)
C
Figure I
Evidence for recombination*
664J
...*
Trisomy
Yes
No
XXY, XXX
22
9
16
14 (39%)
0 (0%)
12 (43%)
16
21
9.iw
a
*In this analysis, we considered only those trisomies which were
informative for recombination at four or more non-centromeric loci.
Trisomies were scored as positive for recombination if at least one
crossover was detected and negative if informative loci were nonreduced. Data on XXYs and XXXs are from Morton et al'2 and
unpublished observations; data on trisomy 21 are from unpublished
observations.
op.
...
gm:
_nA
4_
_-.
a
4
_4
'.
w
so
maternal meiosis I errors, but in most of these there
only one or two informative loci.
were
Discussion
Trisomy 16 is unusual in at least two respects: its
incidence and its relationship to maternal age. It is
extraordinarily common, occurring in 7 to 8% of all
spontaneous abortions and, by extrapolation, over 1%
of all clinically recognised human pregnancies.' The
reason for this high frequency is unknown, but it is
more likely to result from variation in non-disjunctional
frequency than differential in utero selection among
trisomies. That is,
Fiure 2 Analysis of the meiotic stage of chromosome 16 nondisunction. DNA from two trisomy 16 spontaneous abortims
(SSS.0 and S343.0), knoumfrom other markers to be maternally
derived trisomies, and their parents were digested with EcoRV
and probed with pSE16-2, which detects a satellite variation at
the centromere of chromosome 16.6 Eachfetus has inherited a
subset of the father's restricion fragments but all ofthe mothes
restriction fragments. Assuming the mother to be heterozygous,
the results of the centromere marker studies indicate that
non-dsunction occrred at maternal meiosis I.
trisomy
16 is identified
more
frequently than are trisomies 13, 18, 21, or the sex
chromosome trisomies, despite the fact that the latter
trisomies are compatible with livebirth. Furthermore,
even among spontaneous abortions, trisomy 16 is not
characteristically associated with late fetal wastage.
Thus, it seems likely that, at the time of conception,
trisomy 16 is disproportionately represented. This
implies that there are mechanisms of non-disjunction
largely, or wholly, restricted to chromosome 16, and
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162
Hassold, Pettay, Freeman, Grantham, Takaesu
this is consistent with data on maternal age. Risch et unlikely that failure to recombine is as important in
a18 have shown that trisomy 16 has a completely (log) the genesis of trisomy 16 as it is in the genesis of other
linear increase in frequency with age, with no trisomies.
Alternatively, there may be an association between
evidence for a maternal age independent component
and this has been confirmed by Morton et al.9 In this increased recombination and non-disjunction of
respect, trisomy 16 is unlike other trisomies involving
similar sized chromosomes. From this, Risch et al8
suggested that there may be a single underlying
mechanism of origin for trisomy 16 which is different
from the mechanisms associated with trisomies for the
other small chromosomes.
The results of the present study support this
interpretation. In each of 22 cases of trisomy 16 in
which we were able to determine both the parent and
meiotic stage of origin, the additional chromosome
resulted from non-disjunction at maternal meiosis I.
In four other cases in which we were unable to specify
the parent or stage of origin or both, our observations
were consistent with a maternal meiosis I error. Thus,
in all 26 cases our results were compatible with
maternal meiosis I non-disjunction. Maternal errors
predominate in other trisomies that have been studied
with DNA markers,2 10 11 but to our knowledge no
other trisomy has as high a proportion of cases of
maternal origin. Thus, it may be that the high
incidence of trisomy 16 is attributable to an abnormal
process acting at maternal meiosis I which is more
likely to disrupt disjunction of chromosome 16 than
other chromosomes.
In the second aspect of our study, we were
interested in determining whether or not chromocome
16 non-disjunction might be related to abnormally
low or high levels of recombination. Recent studies
from our laboratory and others suggested an association
between failure to recombine and maternal meiosis I
non-disjunction for the XX'2 and chromosome 21
bivalents'3 (Hassold, unpublished observations).
Therefore, we examined our cases for possible
pairing/recombination failure, but found little evidence
for such an effect. We identified recombinants in each
of the nine cases having four or more informative,
non-centromeric loci; overall, we identified recombinants in 17 of the 22 cases attributable to maternal
meiosis I non-disjunction. This contrasts markedly
with our observations for maternally derived sex
chromosome trisomies and trisomy 21, in which a
significant proportion of cases have no detectable
recombination between the non-disjoined chromosomes (table 2). It may be possible to explain the
difference between trisomies 16 and 21 by invoking
the different genetic lengths of the two chromosomes;
that is, since the chromosome 21 map is smaller we
would expect a higher proportion of apparent nonrecombinants for trisomy 21 than trisomy 16. However, this cannot explain the difference between
trisomy 16 and the sex chromosome trisomies, since
the female X chromosome map is at least as long as
the map for chromosome 16.14 Therefore, it seems
chromosome 16. This has been suggested for Droso-
phila'5 and we have preliminary data linking X
chromosome pericentromeric recombination with
maternal X chromosome non-disiunction. 12 Un-
fortunately, it is not yet possible to evaluate this
hypothesis for trisomy 16; our series is still small and
there are no data from conventional linkage studies on
chromosome 16 pericentromeric recombination.
Nevertheless, our preliminary results already indicate
that, if aberrant recombination is associated with
chromosome 16 non-disjunction, it is likely to be
hyper- and not hyporecombination.
We gratefully acknowledge the assistance of the staff
of the Department of Pathology and the Department
of Obstetrics and Gynecology, Northside Hospital,
Atlanta, GA, and especially Dr Raphael Graves, Dr
Alan Joffe, Dr Gerry Sotomayer, and Chris Wylie.
Additionally, we wish to thank Dr H Willard for
donation of the probe pSE 16-2, and for his comments
on the manuscript. This work was supported by NIH
grants HD 21341 and HD 25509.
1 Hassold T, Jacobs PA. Trisomy in man. Annu Rev Genet
1984;18:69-97.
2 Hassold T, Takaesu N. Analysis of non-disjunction in human
trisomic spontaneous abortions. In: Hassold TJ, Epstein CJ,
eds. Molecular and cytogenetic studies of non-disjunction. New
York: Alan R Liss, 1989:115-34.
3 Hassold T, Chen N, Funkhouser J, et al. A cytogenetic study of
1,000 spontaneous abortions. Ann HuAGenet 1980;44:151-78.
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5 Kidd KK, Bowcock AM, Schmidtke J, et al. Report of the DNA
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6 Greig GM, England SB, Bedford HM, Willard HF. Chromosome-specific alpha satellite DNA from the centromere of
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12 Morton NE, Keats BJ, Jacobs PA, et al. A centromere map of the
X chromosome from trisomies of maternal origin. Ann Hum
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13 Warren AC, Chakravarti A, Wong C, et al. Evidence for reduced
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Molecular studies of
non-disjunction in trisomy 16.
T J Hassold, D Pettay, S B Freeman, M Grantham and N
Takaesu
J Med Genet 1991 28: 159-162
doi: 10.1136/jmg.28.3.159
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